1. Field Of The Invention
The invention relates to a microbiological process for the oxidation of methyl groups on aromatic 5- or 6-ring heterocycles to the corresponding carboxylic acid, with the heterocycle exhibiting no substituent on the carbon atom adjacent to the methyl group to be oxidized.
2. Background Art
These carboxylic acid derivatives can be used, for example, as intermediate products for other chemical syntheses. For example, 2-pyrazinecarboxylic acid is an important intermediate product for the production of the tuberculostatic pyrazinamide (2-pyrazinecarboxylic acid amide) [Roemps Chemie Lexikon, vol. 8, No. 5, (1987), p. 3411].
Thorough studies on the microbiological production of carboxylic acids up to now were conducted with aromatic hydrocarbons. The production of carboxylic acids by microbiological oxidation of methylated aromatic compounds was described in detail in the works of Raymond et al. [Raymond et al., Process Biochem., (1969), pages 71 to 74]. U.S. Pat. No. 3,383,289 describes a process for the biochemical oxidation of methyl groups in aromatic hydrocarbons with a gram-positive microorganism strain of the genus Nocardia. Disadvantages of these processes include, for example, in the methyl group oxidation of aromatic hydrocarbons, the benzene ring of the corresponding acid being cleaved.
In regards to Pseudomonas putida ATCC 33015 it is known that the biochemical oxidation of the methyl groups are toluene to benzoic acid taking place in three steps. By the action of the toluene monoxygenase, benzyl alcohol first results which then in two other steps, that is, catalyzed by an alcohol dehydrogenase and an aldehyde dehydrogenase, is converted to the acid.
In this strain both the Xyl genes, which code for enzymes of the xylene decomposition, and the genes, which are responsible for the regulation of the Xyl genes, are on the plasmid pWWO. This archetypical Tol plasmid has already been thoroughly investigated in a molecular biological manner [Harayama et al., J. Bacteriol., 171, (1989), pages 5048 to 5055; Burlage et al., Appl. Environ. Microbiol. 55, (1989), pages 1323 to 1328].
Also microbiological processes for the oxidation of methyl groups of an N-heterocycle are known from the literature. According to Soviet Union Patent No. 417,468, 2-methylpyridine is oxidized with a gram-positive microorganism strain of the genus Nocardia to the corresponding acid.
Soviet Union Patent No. 228,688 describes a microbiological process for the production of nicotinic acid from 3-methylpyridine with a gram-positive microorganism of the genus Mycobacterium. A microbiological process for the production of nicotinic acid with gram-positive bacteria of the genus Nocardia is known from Soviet Union Patent No. 302,341.
The disadvantages of methyl group oxidation of N-heterocycles with gram-positive bacteria are that with these alkane-utilizing bacteria the mixture ratio of the alkane to the substance to be oxidized has to be precisely adjusted to achieve a biotransformation, and that no biotransformation of the substance takes place in the absence of the alkane, i.e., the alkane used for the induction always has to be present even in the reaction of the substrate. By comparison tests with the gram-positive bacterium Nocardia and gram-negative Pseudomonas putida ATCC 330of applicant, it was possible to show clearly that Nocardia even in the presence of an alkane, such as, dodecane, does not oxidize 3-methylpyridine to the corresponding nicotinic acid.
Further, U.S. Pat. No. 4,859,592 describes a process for the production of picolinic acid with Pseudomonas putida, an alkyl-substituted aromatic hydrocarbon being formed in the presence of molecular oxygen in a first step by a dioxygenase. The resultant 2-hydroxymuconic acid semialdehyde then being reacted in a second step with ammonia or a primary amine to the corresponding nicotinic acid. The disadvantage of this process is that the corresponding picolinic acid is formed only in the second step by the reaction of the 2-hydroxymuconic acid semialdehyde with ammonia.
A microbiological process for the oxidation of methyl groups on heterocycles is also described in commonly-owned U.S. Ser. No. 650,589, filed on Feb. 5, 1991, now U.S. Pat. No. 5,104,798, issued on Apr. 14, 1992, (Swiss Patent Application No. 458/90, filed on Feb. 13, 1990), (both of which are not prior art). In this process, the Pseudomonas microorganisms are cultured first in a culture medium, for example, with p-xylene as the sole carbon source and energy source, are afterward separated and then the biotransformation is performed by the addition of the feedstock. By exhaustion of the biomass activity, relative small product concentrations are achieved in this two-step process.